A high diversity of culturable foliar endophytic fungi is known from various mangrove plants, and the core taxa include species from Colletotrichum, Pestalotiopsis, Phoma, Phomopsis, Sporomiella, among others. Since a small fraction of fungi is able to grow in culture, this study investigated the diversity of fungi associated with leaves of Acanthus ilicifolius var. xiamenensis using both isolation and metabarcoding approaches. A total of 203 isolates were cultured from surface-sterilized leaves, representing 47 different fungal species: 30 species from the winter samples (104 isolates), and 26 species from the summer samples (99 isolates). Ascomycota was dominant in both types of leaf samples, while Basidiomycota was isolated only from the summer samples. Drechslera dematioidea (10.58%, percentage of occurrence), Colletotrichum sp. 3 (7.69%) and Alternaria sp. (7.69%) were dominant in the winter samples; Fusarium oxysporum (13.13%), Diaporthe endophytica (10.10%) and Colletotrichum sp. 1 (9.09%) in the summer samples. Overall, Corynespora cassiicola (6.90%), F. oxysporum (6.40%) and Guignardia sp. (6.40%) had the highest overall percentage of occurrence. In the metabarcoding analysis, a total of 111 operational taxonomic units (OTUs) were identified from 17 leaf samples: 96 OTUs from the winter and 70 OTUs from the summer samples. Sequences belonging to Ascomycota and Basidiomycota were detected in both samples but the former phylum was dominant over the latter. Based on read abundance, taxa having the highest percentage of occurrence included Alternaria sp. (3.46%), Cladosporium delicatulum (2.56%) and Pyrenochaetopsis leptospora (1.41%) in the winter leaves, and Aureobasidium sp. (10.72%), Cladosporium sp. (7.90%), C. delicatulum (3.45%) and Hortaea werneckii (3.21%) in the summer leaves. These latter four species also had the highest overall percentage of occurrence. Combining the results from both methods, a high diversity of fungi (at least 110 species) was found associated with leaves of A. ilicifolius var. xiamenensis. Many of the fungi identified were plant pathogens and may eventually cause diseases in the host.

BackgroundAcanthus ilicifolius var. xiamenensis (Acanthaceae) is an old world mangrove species and has long been used as a folk remedy for treating various ailments in traditional medicine. The nature source of A. ilicifolius var. xiamenensis is now in short supply because of the urban development and habitat destruction. To better utilize this resource, biodiversity and bioactivity of endophytic fungi isolated from A. ilicifolius var. xiamenensis were investigated.ResultsA total of 168 fungal isolates were cultured from leaves and stems of the mangrove plant collected in January (winter) and July (summer) 2014 at Kinmen County, Taiwan. Spent culture extract of 28 isolates were found to have bioactivities against one of the following pathogenic microorganisms: the bacteria Bacillus subtilis, Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative) and the fungi Candida albicans and Cryptococcus neoformans. These positive extracts were mostly active against the Gram-positive bacteria and C. albicans. Corynespora cassiicola NTOU4889 and Xylaria sp. NTOU4900 inhibited growth of all 3 test bacteria whereas Phellinus noxius NTOU4917 inhibited both test fungi. A further anti-inflammatory study of culture extracts of these 28 isolates revealed that extracts with a high iNOS inhibition caused a low viability of cells, and those with a low iNOS inhibition had a high cell viability. Three extracts showed low cytotoxicity (i.e. > 100% cell viability) and high iNOS inhibition (< 15% of NO production) of cells and they were Phoma sp. 2 NTOU4338, Nodulisporium sp. NTOU4868 and Guignardia sp. NTOU4871.ConclusionThese results indicate that the endophytic fungi associated with A. ilicifolius var. xiamenensis can be a potential source of novel natural active substance.

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